Characterization of Liver Function in Transdifferentiated Hepatocytes

Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom.
Journal of Cellular Physiology (Impact Factor: 3.84). 01/2006; 206(1):147-59. DOI: 10.1002/jcp.20438
Source: PubMed


We previously demonstrated that dexamethasone (Dex) induces the transdifferentiation (or conversion) of the pancreatic progenitor cell line AR42J-B13 (B13) to hepatocytes based on the expression of liver proteins. We have extended our original observations to determine: (1) the effects of Dex on pancreatic gene expression; (2) the time course of expression of liver enriched transcription factors during conversion from pancreatic to hepatic phenotype; (3) the functional potential of transdifferentiated hepatocytes; (4) the proliferative capacity of transdifferentiated hepatocytes; and (5) whether ectopic expression of transcription factors can induce the hepatic phenotype in pancreatic B13 cells. The results were as follows. The B13 cell markers amylase, synaptophysin, and neurofilament were lost in transdifferentiated hepatocytes compared to control cells and the liver enriched transcription factors C/EBPbeta and C/EBPalpha were induced first, followed by HNF4alpha and then RXRalpha. Using RT-PCR analysis and immunolocalisation studies, we detected hepatic markers (e.g., apolipoprotein B) in Dex-treated cells. In transdifferentiated hepatocytes albumin was secreted, insulin stimulated lipid deposition and ciprofibrate enhanced the expression of catalase. Proliferation of transdifferentiated hepatocytes is promoted in the presence of HGF and NEAA as indicated by the co-expression of the cell cycle markers cyclin D and phosphohistone H3 with liver proteins. Lastly, ectopic expression of C/EBPalpha or C/EBPbeta in AR42J-B13 cells was sufficient to induce transdifferentiation, based on nuclear localization of HNF4alpha and induction of UDP-glucuronosyltransferase expression. These results indicate that the B13 progenitor cell model is suitable for studying liver function and for understanding the molecular and cellular events that occur during transdifferentiation.

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Available from: Chia-Ning Shen, Jan 27, 2015
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    • "Functional lineage conversion without complete erasure of parental gene expression is not uncommon. For example, when hepatocyte cells trans-differentiate along the pancreatic lineage, they express insulin yet retaining some expression of hepatocyte-specific genes [61]. Similarly, in the case of retrograde reprogramming of somatic cells toward pluripotency by forced expression of transcription factors, residual expression of parental genes and epigenetic signatures persist and are carried over to differentiated lineages [62,63]. "
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    ABSTRACT: The neural stem cells discovered in the adult ciliary epithelium (CE) in higher vertebrates have emerged as an accessible source of retinal progenitors; these cells can self-renew and possess retinal potential. However, recent studies have cast doubt as to whether these cells could generate functional neurons and differentiate along the retinal lineage. Here, we have systematically examined the pan neural and retinal potential of CE stem cells. Molecular and cellular analysis was carried out to examine the plasticity of CE stem cells, obtained from mice expressing green fluorescent protein (GFP) under the influence of the promoter of the rod photoreceptor-specific gene, Nrl, using the neurospheres assay. Differentiation was induced by specific culture conditions and evaluated by both transcripts and protein levels of lineage-specific regulators and markers. Temporal pattern of their levels were examined to determine the expression of genes and proteins underlying the regulatory hierarchy of cells specific differentiation in vitro. Functional attributes of differentiation were examined by the presence of current profiles and pharmacological mobilization of intracellular calcium using whole cell recordings and Fura-based calcium imaging, respectively. We demonstrate that stem cells in adult CE not only have the capacity to generate functional neurons, acquiring the expression of sodium and potassium channels, but also respond to specific cues in culture and preferentially differentiate along the lineages of retinal ganglion cells (RGCs) and rod photoreceptors, the early and late born retinal neurons, respectively. The retinal differentiation of CE stem cells was characterized by the temporal acquisition of the expression of the regulators of RGCs and rod photoreceptors, followed by the display of cell type-specific mature markers and mobilization of intracellular calcium. Our study demonstrates the bonafide retinal potential of adult CE stem cells and suggests that their plasticity could be harnessed for clinical purposes once barriers associated with any lineage conversion, i.e., low efficiency and fidelity is overcome through the identification of conducive culture conditions.
    BMC Neuroscience 10/2013; 14(1):130. DOI:10.1186/1471-2202-14-130 · 2.67 Impact Factor
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    • "Although enhanced activity of C/EBPß can promote differentiation of normal and malignant myeloid precursors, a complete loss of C/EBPß activity might be necessary to disrupt the differentiation potential of CML-BC (chronic myelogenous leukemia blast crisis) progenitors [35]. Using the rat pancreatic cell line AR42J-B13, researchers showed that transfection with C/EBP-βprovoked hepatic differentiation [36], while transfection with a dominant negative inhibitor of C/EBP-β will both inhibit glucocorticoid-induced formation of hepatocytes and also caused loss of the hepatic phenotype from cells maintained for a long periods in dexamethasone [37]–[39]. C/EBPß is also activated in organ cultures of embryonic pancreas following glucocorticoid treatment and its expression is associated with hepatic differentiation [37], [40]. Our results indicated that miR-92b played the role most probably by influencing C/EBPß. "
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    ABSTRACT: microRNAs (miRNAs) are short noncoding RNAs that negatively regulate gene expression. Although recent evidences have been indicated that their aberrant expression may play an important role in cancer stem cells, the mechanism of their deregulation in neoplastic transformation of liver cancer stem cells (LCSCs) has not been explored. In our study, the HCC model was established in F344 rats by DEN induction. The EpCAM(+) cells were sorted out from unfractionated fetal liver cells and liver cancer cells using the FACS analysis and miRNA expression profiles of two groups were screened through microarray platform. Gain-of-function studies were performed in vitro and in vivo to determine the role of miR-92b on proliferation and differentiation of the hepatic progenitors. In addition, luciferase reporter system and gene function analysis were used to predict miR-92b target. we found that miR-92b was highly downregulated in EpCAM(+) fetal liver cells in expression profiling studies. RT-PCR analysis demonstrated reverse correlation between miR-92b expression and differentiation degree in human HCC samples. Overexpression of miR-92b in EpCAM(+) fetal liver cells significantly increased proliferation and inhibited differentiation as well as in vitro and in vivo studies. Moreover, we verified that C/EBPß is a direct target of miR-92b and contributes to its effects on proliferation and differentiation. We conclude that aberrant expression of miR-92b can result in proliferation increase and differentiation arrest of hepatic progenitors by targeting C/EBPß.
    PLoS ONE 08/2013; 8(8):e68004. DOI:10.1371/journal.pone.0068004 · 3.23 Impact Factor
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    • "The other factors with a similar genomic signature to Pdx1 are all known to play a role in pancreas development, including Nkx6-1 which promotes Pdx1-induced liver to pancreas transdifferentiation [23]. An apparent outlier to our expected pattern is Cebpb, which by itself can convert a pancreatic progenitor cell line to hepatocyte-like cells [22], but is only slightly more expressed in liver cells and slightly more repressed in pancreatic islet cells. Although Cebpb is typically considered a hepatic TF, it also expresses in pancreatic beta-cells, particularly under metabolic stress [24]. "
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    ABSTRACT: Transcription factors that can convert adult cells of one type to another are usually discovered empirically by testing factors with a known developmental role in the target cell. Here we show that standard genomic methods (RNA-seq and ChIP-seq) can help identify these factors, as most are more strongly Polycomb repressed in the source cell and more highly expressed in the target cell. This criterion is an effective genome-wide screen that significantly enriches for factors that can transdifferentiate several mammalian cell types including neural stem cells, neurons, pancreatic islets, and hepatocytes. These results suggest that barriers between adult cell types, as depicted in Waddington's "epigenetic landscape", consist in part of differentially Polycomb-repressed transcription factors. This genomic model of cell identity helps rationalize a growing number of transdifferentiation protocols and may help facilitate the engineering of cell identity for regenerative medicine.
    PLoS ONE 05/2013; 8(5):e63407. DOI:10.1371/journal.pone.0063407 · 3.23 Impact Factor
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